The cellular ESCRT pathway mediates budding of many enveloped viruses, including HIV-1. This pathway also catalyzes analogous membrane fission reactions during intraluminal endosomal vesicle formation and the abscission stage of cytokinesis. In the case of HIV-1, late domain motifs within the viral p6Gag protein bind and recruit two early-acting ESCRT factors, ESCRT-I and ALIX. These factors, in turn, recruit the ESCRT-III and VPS4 complexes, which then collaborate to catalyze membrane fission. During the current funding cycle, we have added to our understanding of HIV-1 budding by: 1) identifying the MVB12 class of ESCRT-I subunits, 2) determining structures of ALIX, both free and in complex with its binding sites on viral Gag and CHMP4(ESCRT-III) proteins, 3) determining structures of ESCRT-III proteins and characterizing their mechanism of autoinhibition, 4) identifying CHMP2 and CHMP4 as the key ESCRT-III subunits in HIV-1 budding, 5) demonstrating that most ESCRT-III proteins can polymerize into helical tubes and filaments, and 6) showing how VPS4 enzymes bind their ESCRT-III substrates and assemble into dodecameric complexes comprising two stacked hexameric rings. During the next funding cycle, we will study how core ESCRT factors form higher-order assemblies that work together to mediate HIV-1 budding. Experiments in Aim1 will focus on characterizing the ESCRT-III/VPS4 membrane fission machinery. Specifically, we will determine how ESCRT-III proteins interact, co-polymerize, and function in virus budding. We will also test whether Vps4 enzymes disassemble ESCRT-III filaments by unfolding individual subunits, translocating them through the central pore of the dodecamer, and releasing them back into the cytoplasm. Experiments in Aim2 will focus on elucidating the regulatory interactions that allow ESCRT factors to co-assemble rapidly, accurately, and reversibly at sites of virus budding. Specifically, we will define the conformational changes required for ALIX activation, test whether ALIX and ESCRT-I function together as a higher-order supercomplex, and determine how the CHMP5-LIP5 complex activates the VPS4 ATPase. In Aim3, we will attempt to reconstitute HIV-1 budding in vitro. Our strategy will be to assemble pure myristoylated HIV-1 Gag-nucleic acid complexes on giant unilamellar vesicles (GUVs) and then use a minimal set of pure recombinant human ESCRT proteins to catalyze membrane fission and release virus-like particles into GUV interiors. Toward this end, we have defined the minimal ESCRT machinery required for HIV-1 budding, developed methods for producing recombinant myristoylated HIV-1 Gag (Myr-Gag) and all of the essential human ESCRT factors, and shown that Myr-Gag-nucleic acid complexes assemble into punctate complexes on GUV surfaces that contain PI(4,5)P2. These experiments will test our ability to reconstitute HIV budding from pure components and provide powerful systems for analyzing HIV assembly and budding in mechanistic detail.